Komagataella phaffii
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| Komagataella phaffii | ||||||||||||
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Komagataella phaffii is a type of yeast which, under the name Pichia pastoris, represents one of the most important expression systems in biotechnology . At the same time, K. phaffii is an important model organism for research. K. phaffii is one of the methylotrophic yeasts and is therefore ableto utilize methanol as the only source of energy and carbon.
Surname
The strains used today in biotechnology were originally incorrectly classified as Pichia pastoris yeast and accordingly sold commercially. In the course of a reclassification, the species Pichia pastoris was assigned to the genus Komagataella in 1995 and renamed Komagataella pastoris . Using rRNA sequence analyzes in 2009, it was possible to show that all Pichia pastoris strains used in biotechnology are not Komagataella pastoris , but the closely related yeast species Komagataella phaffii . In biotechnology, however, the name Pichia pastoris is still used for the expression system.
Pichia pastoris in biotechnology
P. pastoris was first cultivated on a commercial scale in the 1970s by the Phillips Petroleum Company (today: ConocoPhillips ) and sold directly as protein-containing animal feed . The interest in the yeast was mainly aroused by its methylotrophic character, since methanol was very cheap compared to other nutrient media . As a result of the 1973 oil crisis , the price of methanol rose significantly while the price of soybeans , the largest alternative source of animal feed, fell. The use of P. pastoris was therefore no longer of economic importance for a long time. It was only through the advancing research in the field of genetics and the development of recombinant DNA technologies ( genetic engineering ) that P. pastoris became increasingly popular as an expression system. Since the yeast received the so-called GRAS status ( generally recognized as safe ; generally classified as safe) by the US regulatory authority , the Food and Drug Administration (FDA), it has also established itself for the production of active pharmaceutical ingredients. Among other things, this is due to the fact that disease-causing factors such as pyrogens and lysogenic viruses do not exist in yeast.
Currently more than 500 recombinant proteins (including some pharmaceutical products) are cloned and produced in P. pastoris . The following table gives some examples for the expression of proteins foreign to the organism in P. pastoris :
| organism | protein | Function / effect |
|---|---|---|
| Mammal / human | Anti-HBs Fab fragment | Prevention and treatment against hepatitis B virus |
| Mammal / human | Dopamine D2S receptor | G protein |
| Mammal / human | DNA - topoisomerase I (hTopo I) | DNA replication , transcription and recombination |
| Clostridium botulinum | Clostridium botulinum serotype F | antigen |
| Escherichia coli | Phytase | Use as a supplement for animal feed |
| Thermus aquaticus | YT-1 aqualysin I. | Heat stable serine protease |
| Rhizopus oryzae | Lipase | Catalytic cleavage of triacylglycerides |
| Trametes versicolor | Laccase (lcc1) | Phenol oxidase |
| Candida antarctica | CBM-CALB fusion protein | Lipase |
| Hevea brasiliensis | Hydroxynitrile lyase | Industrial enzymes for biocatalysis |
| Amaryllidaceae | Snowdrop agglutinin | Binding of D-mannose units |
| Panax ginseng | Gsp ( Panax ginseng C) | Potential active ingredient against diabetes |
| Oryza sativa | Alpha-amylase (Amy 1A / 3D) | Starch hydrolysis |
| Chondrosia reniformis | Prolyl 4-hydroxylase (P4H) | Hydroxylation of proline in collagen |
The Pichia pastoris expression system
The utilization of methanol requires the presence of proteins , which alcohol can smuggle into the yeast metabolism. In P. pastoris these include alcohol oxidases I and II (AoxI, AoxII), which are the initial enzymes that oxidize methanol to hydrogen peroxide and formaldehyde . But alcohol oxidase I in particular has a very low affinity for oxygen; the yeast compensates for this deficiency by producing large amounts of AoxI (up to 30% of the cell protein). To make this possible, the subject AOXI - gene the control of very strong AOX - promoter , which only in the presence of methanol to transcription leads of the corresponding gene. This results in a promoter that can be induced by adding methanol. If the gene sequence of a recombinant protein is now under the control of an AOX promoter (usually AOX1 ), the accumulation of biomass can be separated from the actual protein production in a fermentation process , which is usually the aim. Only when methanol is added as an inducer is the AOX promoter read and thus the recombinant protein is also synthesized. P. pastoris can be grown over a wide pH range without significantly affecting growth. In contrast to some other yeasts such as Saccharomyces cerevisiae , Pichia pastoris can be cultivated to very high cell densities. Post-translational modifications such as the formation of disulfide bridges or the glycosylation of proteins (the attachment of specific sugar residues) are carried out by the yeast. Pichia can also secrete proteins very efficiently and therefore channel them out of the cell, which facilitates subsequent processing steps ( downstream processing ). In P. pastoris coming for use vectors , which are those genetic constructs that carry the recombinant protein are usually integrated into the host genome.
Glycosylation and " Humanization "
The glycosylation mechanism of yeasts in particular is a recurring problem in the expression of human, pharmaceutical active ingredients. Usually proteins produced in yeast are hypermannosylated, therefore up to 200 mannose residues are added to them (in P. pastoris : 8-14 mannose residues) . In each case, these structures differ significantly from human glycosylation, which is of a more complex type. So-called hypermannosylated proteins are partially inoperable in the human organism, can trigger allergic reactions and have a very short half-life in human serum because they are mainly bound by mannose receptors in the liver. To fully glycosylated human proteins in a high-density fermentation with P. pastoris to manufacture, has in recent years on the " Humanization research" (humanization) of glycosylation. In principle, unwanted yeast metabolism enzymes that are responsible for hypermannosylation, for example, are removed, while genes that are involved in human glycosylation are introduced into the organism using recombinant DNA techniques. In 2004, the human protein erythropoietin (EPO) in its natural, human glycosylation could be produced in a P. pastoris strain for the first time . The aim of further research in this area is to apply the results to other expression systems and to increase expression rates.
literature
- Cregg J. (2007): Pichia Protocols (Methods in Molecular Biology). Humana Press; 2nd edition ISBN 978-1-58829-429-6
- Gellissen G. (Ed): (2005) Production of recombinant proteins - novel microbial and eukaryotic expression systems. Wiley-VCH, Weinheim ISBN 978-3-527-31036-4
Individual evidence
- ↑ Y. Yamada, M. Matsuda, K. Maeda, K. Mikata: The phylogenetic relationships of methanol-assimilating yeasts based on the partial sequences of 18S and 26S ribosomal RNAs: the proposal of Komagataella gen. Nov. (Saccharomycetaceae). In: Bioscience, biotechnology, and biochemistry. Volume 59, Number 3, March 1995, pp. 439-444, doi : 10.1271 / bbb.59.439 , PMID 7766181 .
- ↑ CP Kurtzman: Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. In: Journal of industrial microbiology & biotechnology. Volume 36, Number 11, November 2009, pp. 1435-1438, doi : 10.1007 / s10295-009-0638-4 , PMID 19760441 .
- ↑ Cereghino JL, Cregg JM; Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews 24 (2000) 45-66
- ↑ Macauley-Patrick S., Fazenda ML, McNeil B., Harvey LM; Heterologous protein production using the Pichia pastoris expression system. Yeast (2005); 22: 249-270.
- ↑ a b Marina Pozzolini, Sonia Scarfi, Francesca Mussino, Annalisa Salis, Gianluca Damonte, Umberto Benatti, Marco Giovine: Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen . Journal of Biotechnology 208, Aug. 20, 2015; Pp. 28-36. doi : 10.1016 / j.jbiotec.2015.05.007
- ↑ http://www.microbialcellfactories.com/content/5/1/17
- ↑ Neta D .; Asparagine-linked glycosylation in the yeast Golgi. Biochimica et Biophysica Acta 1426 (1999) 309-322.
- ^ Hamilton SR, Gerngross TU Glycosylation engineering in yeast: the advent of fully humanized yeast. Current Opinion in Biotechnology 2007, 18: 1-6.
- ↑ Bretthauer RK; Genetic engineering of Pichia pastoris to humanize N-glycosylation of proteins. Trends in Biotechnology 2003, 21: 459-462.
- ↑ Hamilton SR, Bobrowicz P., Bobrowicz B., Davidson RC, Li H., Mitchell T., Nett JH, Rausch S., Stadheim TA, Wischnewski H .; Production of complex human glycoproteins in yeast. Science 2003, 301: 1244-1246.